Hematopoietic Stem Cells Can Be Enriched

I. L. Weissman and colleagues developed a novel way of enriching the concentration of mouse hematopoietic stem cells, which normally constitute less than 0.05% of all bone-marrow cells in mice. Their approach relied on the use of antibodies specific for molecules known as differentiation antigens, which are expressed only by particular cell types. They exposed bone-marrow samples to antibodies that had been labeled with a fluorescent compound and were specific for the differentiation antigens expressed on the surface of mature red and white blood cells (Figure 2-6). The labeled cells were then removed by flow cytometry with a fluorescence-activated cell sorter (see Chapter 6). After each sorting, the remaining cells were assayed to determine the number needed for restoration of hematopoiesis in a lethally x-irradiated mouse. As the pluripotent stem cells were becoming relatively more numerous in the remaining population, fewer and fewer cells were needed to restore hematopoiesis in this system. Because stem cells do not express differentiation antigens

Antigen

Antigen

Cytokine receptor

Activated B cell

Cytokine receptor

Activated B cell

Apoptotic cell

Plasma cell

B memory cell

Apoptotic cell

Plasma cell

B memory cell

FIGURE 2-5

Regulation of activated B-cell numbers by apoptosis. Activation of B cells induces increased expression of cytokine receptors and decreased expression of Bcl-2. Because Bcl-2 prevents apoptosis, its reduced level in activated B cells is an important factor in making activated B cells more susceptible to programmed cell death than either naive or memory B cells. A reduction in activating signals quickly leads to destruction of excess activated B cells by apoptosis. Similar processes occur in T cells.

Hematopoiesis From Pluripotent
1 X 103 partly enriched cells
Hematopoiesis Mouse
Restore hematopoiesis, mouse lives

30-100 fully enriched cells

30-100 fully enriched cells

Restore hematopoiesis, mouse lives

React with Fl-antibodies against Sca-1

Stem Progenitor cell cells

Differentiated cells

FIGURE 2-6

Enrichment of the pluripotent stem cells from bone marrow. (a) Differentiated hematopoietic cells (white) are removed by treatment with fluorescently labeled antibodies (Fl-antibodies) specific for membrane molecules expressed on differentiated lineages but absent from the undifferentiated stem cells (S) and progenitor cells (P). Treatment of the resulting partly enriched preparation with antibody specific for Sca-1, an early differentiation antigen, removed most of the progenitor cells. M = monocyte; B = basophil; N = neutrophil; Eo = eosinophil; L = lymphocyte; E = erythrocyte. (b) Enrichment of stem-cell preparations is measured by their ability to restore hematopoiesis in lethally irradiated mice. Only animals in which hematopoiesis occurs survive. Progressive enrichment of stem cells is indicated by the decrease in the number of injected cells needed to restore hematopoiesis. A total enrichment of about 1000fold is possible by this procedure.

known to be on developing and mature hematopoietic cells, by removing those hematopoietic cells that express known differentiation antigens, these investigators were able to obtain a 50- to 200-fold enrichment of pluripotent stem cells. To further enrich the pluripotent stem cells, the remaining cells were incubated with various antibodies raised against cells likely to be in the early stages of hematopoiesis. One of these antibodies recognized a differentiation antigen called stem-cell antigen 1 (Sca-1). Treatment with this antibody aided capture of undifferentiated stem cells and yielded a preparation so enriched in pluripotent stem cells that an aliquot containing only 30-100 cells routinely restored hematopoiesis in a lethally x-irradiated mouse, whereas more than 104 nonenriched bone-marrow cells were needed for restoration. Using a variation of this approach, H. Nakauchi and his colleagues have devised procedures that allow them to show that, in 1 out of 5 lethally irradiated mice, a single hematopoietic cell can give rise to both myeloid and lymphoid lineages (Table 2-3).

It has been found that CD34, a marker found on about 1% of hematopoietic cells, while not actually unique to stem cells, is found on a small population of cells that contains stem cells. By exploiting the association of this marker with stem cell populations, it has become possible to routinely enrich preparations of human stem cells. The administration of human-cell populations suitably enriched for CD34+ cells

TABLE 2-3

Reconstitution of hematopoeisis by HSCs

Number of enriched HSCs

Number of mice reconstituted (%)

2 5 of 21 (23.8%) 5 9 of 17 (52.9%) 10 10 of 11 (90.9%) 20 4 of 4 (100%)

SOURCE: Adapted from M. Osawa, et al. 1996. Science 273:242.

(the " + " indicates that the factor is present on the cell membrane) can reconstitute a patient's entire hematopoietic system (see Clinical Focus).

A major tool in studies to identify and characterize the human hematopoietic stem cell is the use of SCID (severe combined immunodeficiency) mice as in vivo assay systems for the presence and function of HSCs. SCID mice do not have B and T lymphocytes and are unable to mount adaptive immune responses such as those that act in the normal rejection of foreign cells, tissues, and organs. Consequently, these animals do not reject transplanted human cell populations containing HSCs or tissues such as thymus and bone marrow. It is necessary to use immunodeficient mice as surrogate or alternative hosts in human stem-cell research because there is no human equivalent of the irradiated mouse. SCID mice implanted with fragments of human thymus and bone marrow support the differentiation of human hematopoietic stem cells into mature hematopoietic cells. Different subpopulations of CD34+ human bone-marrow cells are injected into these SCID-human mice, and the development of various lineages of human cells in the bone-marrow fragment is subsequently assessed. In the absence of human growth factors, only low numbers of granulocyte-macrophage progenitors develop. However, when appropriate cytokines such as erythropoietin and others are administered along with CD34+ cells, progenitor and mature cells of the myeloid, lymphoid, and erythroid lineages develop. This system has enabled the study of subpopulations of CD34+ cells and the effect of human growth factors on the differentiation of various hematopoietic lineages.

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Essentials of Human Physiology

Essentials of Human Physiology

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Responses

  • ruta
    Why irradiation remove memory B cells?
    8 years ago
  • DAWID
    Can be enriched through thymus cell antigen?
    8 years ago
  • teagan
    Can irradiated cells be labelled with antibodies?
    8 years ago

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